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PubMed1997

[Selenoproteins in bone, gastrointestinal tract and thyroid gland of the human].

BASIS: Selenium is an essential trace element, which is incorporated as selenocysteine (secys) into specific proteins in a regulated fashion. In the presence of a hairpin loop structure within the 3' untranslated region of the mRNA the opal stop codon UGA is coding for selenocysteine. Selenoprotein functions are dependent on secys incorporation. Members of the family of deiodinases as well as the family of glutathione peroxidases, selenoprotein P and thioredoxin reductase are selenoproteins. DISCUSSION: Bone, the intestine and the thyroid rely on antioxidant systems against potential cell and DNA damage through endogenous and environmental peroxides and reactive oxygen species (ROS) potentially promoting inflammation and tumorigenesis. Optimized cell defense through antioxidant selenoproteins requires optimal selenium supplementation of the organism. We have analyzed the expression of selenoproteins in these tissues, thus providing molecular tools to further elucidate optimal selenium supply on a cellular level. CONCLUSION: Clinical intervention studies that focus on the development of disease must confirm the relevance of optimized selenium supply for the pathogenesis, prevention and therapy of metabolic bone disease as well as chronic (autoimmune) inflammation and tumorigenesis in the thyroid and intestine.

Bone and Bones↗
PubMed1987

Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite.

The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.

Bacterial Proteins↗
PubMed1999

Molecular evidence for heterogeneity of the multiple-drug-resistant Mycobacterium tuberculosis population in Scotland (1990 to 1997).

Multiple-drug-resistant Mycobacterium tuberculosis (MDR-MTB) has been well studied in hospitals or health care institutions and in human immunodeficiency virus-infected populations. However, the characteristics of MDR-MTB in the community have not been well investigated. An understanding of its prevalence and circulation within the community will help to estimate the problem and optimize the strategies for control and prevention of its development and transmission. In this study, MDR-MTB isolates from Scotland collected between 1990 and 1997 were characterized, along with non-drug-resistant isolates. The results showed that they were genetically diverse, suggesting they were unrelated to each other and had probably evolved independently. Several new alleles of rpoB, katG, and ahpC were identified: rpoB codon 525 (ACC-->AAC; Thr525Asn); katG codon 128 (CGG-->CAG; Arg128Gln) and codon 291 (GCT-->CCT; Ala291Pro); and the ahpC synonymous substitution at codon 6 (ATT-->ATC). One of the MDR-MTB isolates from an Asian patient had an IS6110 restriction fragment length polymorphism pattern very similar to that of the MDR-MTB W strain and had the same drug resistance-related alleles but did not have any epidemiological connection with the W strains. Additionally, a cluster of M. tuberculosis isolates was identified in our collection of 715 clinical isolates; the isolates in this cluster had genetic backgrounds very similar to those of the W strains, one of which had already developed multiple drug resistances. The diverse population of MDR-MTB in Scotland, along with a low incidence of drug-resistant M. tuberculosis, has implications for the control of the organism and prevention of its spread.

Alleles↗
PubMed1988

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

Base Sequence↗
PubMed2026

The proteomic origin of the genetic code.

INTRODUCTION: The origin and evolution of the genetic code is a central problem in molecular biology. Classical models have emphasized stereochemistry, frozen accidents, or adaptive optimization, often treating proteins as passive products of preexisting codes. More recent views instead portray the code as a dynamic, coevolving system shaped by reciprocal interactions among amino acids, RNA, and early catalysts. AREAS COVERED: Here, I review efforts of phylogeny reconstruction of the history of tRNA, protein structural domains, and dipeptide sequences in proteomes. These complementary approaches allow exploration of the entry of amino acids and codons into the code, and the transition from an operational RNA code in the tRNA acceptor arm to the canonical code in the anticodon loop. Evidence for ancestral synthetase enzymes with dual functions in aminoacylation and peptide-bond formation, as well as early bidirectional (sense-antisense) coding reflected in dipeptide-antidipeptide emergence is also discussed. EXPERT OPINION: The genetic code is best viewed as a proteome-driven, evolvable system in which early peptides actively shaped coding rules by stabilizing structure, expanding chemical diversity, and enhancing catalysis. This perspective connects origin-of-life studies with modern efforts of code expansion, translational engineering, and peptide-based therapeutics, highlighting the impact of the code's proteomic origin.

Genetic Code↗
PubMed2006

Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis.

OBJECTIVE: The rat is one of the most important model organisms for biomedical and pharmacological research. However, the generation of novel models for studying specific aspects of human diseases largely depends on selection for specific traits using existing rat strains, thereby solely depending on naturally occurring variation. This study aims to provide the tools to manipulate the rat genome in a more directed way. METHODS: We developed robust, automated, and scaleable reverse genetic methodology based on ENU (N-ethyl-N-nitrosourea)-driven target-selected mutagenesis. Optimal mutagenesis conditions have been determined in three different rat strains and a universal, rapid, and cost-effective dideoxy resequencing-based screening setup was established for mutation discovery. The effectiveness of the approach is illustrated by the identification of 120 induced mutations in a set of genes of interest, including six that result in unique rat knockout models due to the introduction of premature stop codons. In addition, 56 mutations were found that change amino acids, including critical residues in transmembrane domains of receptors and channels. CONCLUSIONS: The approach described here allows for the systematic generation of knockout and protein function altering alleles in the rat. The resulting induced rat models will be powerful tools for studying many aspects of a wide variety of human diseases.

Alkylating Agents↗
PubMed1995

Transcription of the archaeal trkA homolog in Methanosarcina mazei S-6.

Transcription of the archaeal trkA gene homolog in Methanosarcina mazei S-6 was studied at the optimal growth temperature of 37 degrees C and after heat shock at 45 degrees C. Northern (RNA) blotting results (transcript size) and data from primer extension experiments to map the transcription initiation site indicate that trkA is cotranscribed with another gene. The latter, orf11, encodes a protein of 94 amino acids (10,611 Da) and is located upstream of trkA, with which it overlaps: the translation stop codon of orf11, TGA, shares the bases T and G with the translation start codon of trkA, ATG. These genes' transcription was decreased by heat shock to the point of making the transcript undetectable by Northern or dot blotting procedures. orf11 and trkA differ in codon usage patterns, and the proteins coded by them, i.e., Orf11 and TrkA, are dissimilar in amino acid sequence and composition.

Amino Acid Sequence↗
PubMed1989

Effect of deletions in the 5'-noncoding region on the translational efficiency of phosphoglycerate kinase mRNA in yeast.

Deletions of various sizes were introduced into the region of the yeast PGK gene encoding the 5'-nontranslated portion of the phosphoglycerate kinase (PGK) mRNA. The effect of these deletions on the translational efficiency of the mutant transcripts was analysed by assaying the levels of mutant PGK mRNA and PGK protein in cells transformed with the mutant genes. Quantification of transcript levels by either Northern analysis or a reverse transcription assay demonstrated that there were no significant differences in the levels of mutant PGK mRNA between the various mutants. Thus, the leader sequence does not appear to play a role in determining the relatively long half-life of yeast PGK mRNA. Analysis of PGK protein levels in the various mutants revealed no effect when the length of the leader was reduced from 45 to 27 nucleotides (nt). Protein levels dropped by about a factor 2, however, upon a further decrease to 21 nt. Additional shortening did not cause a further dramatic reduction in translational yield. Even an mRNA containing a leader of only 7 nt was still translated at about 50% of the optimal rate. Therefore, while optimal translation of a yeast mRNA requires a leader length of at least some 30 nt, shorter leaders still allow considerable translation to take place.

Base Sequence↗
PubMed2005

Enhancing the efficiency of cell-free protein synthesis through the polymerase-chain-reaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues.

A method for the rapid generation of intact proteins in a cell-free protein synthesis system was developed. The productivity of the recombinant proteins from the polymerase-chain-reaction-amplified templates was enhanced remarkably using an optimized translation enhancer sequence. The extra amino acid residues derived from the translation enhancer sequence were effectively removed by utilizing the appropriate detergent and peptide cleavage enzyme in the reaction mixture. These results demonstrate the versatility of cell-free protein synthesis in providing optimized and customized reaction conditions for the efficient production of the desired proteins.

Cell-Free System↗
PubMed1997

Oncogenic mutations in ras create HLA-A2.1 binding peptides but affect their extracellular antigen processing.

Point mutations in oncogene products such as ras may create neoantigenic determinants recognizable by T lymphocytes as tumor antigens, that could be marshalled to eliminate a tumor by inducing specific cytotoxic T lymphocytes (CTL) with an appropriate vaccine. Peptide-pulsed dendritic cells are a promising new approach to cancer vaccines. For such an approach to work, the determinant must be appropriately processed to the right size fragment and be presented by an appropriate HLA molecule. We have investigated both of these issues for a series of ras codon 12 and 13 point mutations that contain sequences predicted to bind to HLA-A2.1, the most common class I HLA molecule. We find that not only do the different mutations affect binding to HLA-A2.1, but also they affect extracellular antigen processing in two ways: by influencing the trimming of flanking residues from the longer sequence and by influencing the susceptibility of the optimal decamer to further proteolytic degradation. The influence of internal residues on cleavage of flanking residues downstream demonstrates the importance of distant interactions between separated amino acid side chains and/or conformational effects in determining antigen processing. These results may be important in designing an effective vaccine to induce mutant ras-specific tumor immunity.

Antigen Presentation↗
PubMed1996

The promoter and transcriptional unit of a novel herpes simplex virus 1 alpha gene are contained in, and encode a protein in frame with, the open reading frame of the alpha 22 gene.

The herpes simplex virus type 1 genome encodes a set of genes (alpha genes) expressed in the absence of de novo viral protein synthesis. Earlier studies have shown that the product of the alpha 22 gene, a member of this set, is nucleotidylylated by casein kinase II and phosphorylated by viral protein kinases encoded by UL13 and US3. Mutants lacking the carboxyl-terminal domain starting with amino acid 200 exhibit reduced capacity to replicate in primary human cell strains or in cells of rodent derivation and also exhibit reduced expression of a subset of gamma or late genes. We report that the domain of the alpha 22 gene is transcribed by two 3'-coterminal mRNAs. The longer transcript reported encodes the 420-amino-acid alpha 22 protein, whereas the shorter transcript reported here encodes a protein containing the carboxyl-terminal 273 amino acids of the alpha 22 protein. The shorter gene is designated US1.5. The US1.5 mRNA is synthesized in cells infected and maintained in the presence of cycloheximide and under other conditions which restrict viral gene expression to alpha genes. In-frame insertion of linkers encoding 18, 21, or 22 amino acids after codon 200 or 240 of the alpha 22 protein did not affect the known functions or phenotype associated with the wild-type alpha 22 gene or its product. Earlier studies have placed the nucleotidylylated sequences in the amino-terminal portion of the protein. The results of these studies indicate that the US1.5 gene encodes the functions associated with replication in human primary or rodent cells and optimal expression of alpha 0 and gamma genes. This finding brings the number of genes known to map in the unique short region of the herpes simplex virus type 1 DNA to 14 and the total number of different genes to 78.

Amino Acid Sequence↗
PubMedDate not supplied

Distinct stages of protein evolution as suggested by protein sequence analysis.

Evolution of proteins encoded in nucleotide sequences began with the advent of the triplet code. The chronological order of the appearance of amino acids on the evolution scene and the steps in the evolution of the triplet code have been recently reconstructed (Trifonov, 2000b) on the basis of 40 different ranking criteria and hypotheses. According to the consensus chronology, the pair of complementary GGC and GCC codons for the amino acids alanine and glycine appeared first. Other codons appeared as complementary pairs as well, which divided their respective amino acids into two alphabets, encoded by triplets with either central purines or central pyrimidines: G, D, S, E, N, R, K, Q, C, H, Y, and W (Glycine alphabet G) and A, V, P, S, L, T, I, F, and M (Alanine alphabet A). It is speculated that the earliest polypeptide chains were very short, presumably of uniform length, belonging to two alphabet types encoded in the two complementary strands of the earliest mRNA duplexes. After the fusion of the minigenes, a mosaic of the alphabets would form. Traces of the predicted mosaic structure have been, indeed, detected in the protein sequences of complete prokaryotic genomes in the form of weak oscillations with the period 12 residues in the form of alteration of two types of 6 residue long units. The next stage of protein evolution corresponded to the closure of the chains in the loops of the size 25-30 residues (Berezovsky et al., 2000). Autocorrelation analysis of proteins of 23 complete archaebacterial and eubacterial genomes revealed that the preferred distances between valine, alanine, glycine, leucine, and isoleucine along the sequences are in the same range of 25-30 residues, indicating that the loops are primarily closed by hydrophobic interactions between the ends of the loops. The loop closure stage is followed by the formation of typical folds of 100-200 amino acids, via end-to-end fusion of the genes encoding the loop-size chains. This size was apparently dictated by the optimal ring closure for DNA. In both cases the closure into the ring (loop) rendered evolutionarily advantageous stability to the respective structures. Further gene fusions lead to the formation of modern multidomain proteins. Recombinational gene splicing is likely to have appeared after the DNA circularization stage.

Amino Acid Sequence↗
PubMed2002

Requirement for both IL-12 and IFN-gamma signaling pathways in optimal IFN-gamma production by human T cells.

Phytohemagglutinin (PHA)-derived T lymphoblasts or T cell clones from patients genetically deficient in IL-12R beta 1 (IL-12R beta 1(-/-)) or IFN-gamma R1 (IFN-gamma R1(-/-)) produced two- to threefold reduced IFN-gamma levels compared to the corresponding cells from healthy individuals after anti-CD3 and PMA stimulation. Moderate IFN-gamma production was observed in PHA-derived T lymphoblasts or T cell clones derived from healthy subjects in the presence of anti-IFN-gamma R1 or anti-IL-12 mAb, whereas it was negligible in the presence of both mAb. However, when anti-IFN-gamma R1 and/or anti-IL-12 mAb were added during restimulation, the cells produced normal levels of IFN-gamma, indicating that both IFN-gamma and IL-12 had an effect on the priming phase. Moderate production of IFN-gamma was partially enhanced only in IFN-gamma R1(-/-) T cell clones generated in the presence of IL-12, but was almost completely abolished when IL-12R beta 1(-/-) and IFN-gamma R1(-/-) T cell clones were generated in the presence of anti-IFN-gamma R1 or anti-IL-12 mAb, respectively. IL-4 production was enhanced in T cell clones from IL-12R beta 1(-/-),but not from IFN-gamma R1(-/-) patients, whereas IL-10 and IL-2 production did not differ significantly in polyclonal T cells or clones from healthy and deficient individuals. These results indicate that IL-12R beta 1- and IFN-gamma R1-dependent signals co-ordinately regulate IFN-gamma, but not IL-2 and IL-10 production, whereas only IL-12 negatively controls IL-4 production by in vitro-generated T cell clones. Thus, although IL-12 and IFN-gamma signals are each sufficient for moderate production of IFN-gamma by human T cells, both are needed for optimal IFN-gamma production, and in the absence of both IFN-gamma production is completely abrogated.

Antibodies, Monoclonal↗
PubMed2003

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35.

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.

1,4-alpha-Glucan Branching Enzyme↗
PubMed2002

Selenoproteins and selenocysteine insertion system in the model plant cell system, Chlamydomonas reinhardtii.

Known eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants. Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs. Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA. Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas: Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals. These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems. We found that selenium is required for optimal growth of Chlamydomonas: Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals.

Amino Acid Sequence↗
PubMed1988

Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine-plus-cytosine contents.

An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and characterized, we have cloned two additional genes, amyB and amyC, coding for amylases of this thermophile, into Escherichia coli and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coli promoters. Almost all activity was detected in the intracellular fraction in the E. coli cells. The molecular mass and NH2-terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coli transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67,000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59,000). The optimal temperatures for AmyB and AmyC activities on soluble starch were 80 degrees C and 70 degrees C, respectively. Both AmyB and AmyC showed a pH optimum of 5.5. AmyB and AmyC showed a different pattern of starch hydrolysis when examined by thin-layer chromatography. Some homology in the amino acid sequences with the functional regions of Taka-amylase A was found in both AmyB and AmyC. The codon usage in the amyA, amyB and amyC genes was highly biased, which reflects the fact that the guanine-plus-cytosine (G + C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the codons was non-random; the G + C content of the first position of codons is significantly high, whereas that of the third position is somewhat low. In addition, codons consisting only of A and T were preferentially used in this thermophile.

Amino Acid Sequence↗
PubMed1992

Lipofectin enhances cellular uptake of antisense DNA while inhibiting tumor cell growth.

A natural DNA oligomer (15-mer) was synthesized with a sequence complementary to the translation initiation codon region of the human TGF-alpha mRNA and mixed with Lipofectin to form unilamellar complexes. It was found that tumor cell growth was inhibited when HCT116 cells were treated with Lipofectin-DNA oligomer complexes or with Lipofectin alone. Uptake of 32P-labeled 15-mers into colon tumor cells was compared in the presence and absence of Lipofectin. The amount of labeled oligomer found in cells that received optimal ratios of Lipofectin to DNA was 4- to 10-fold higher than the amount found in cells that received 32P-labeled DNA alone. Although Lipofectin-antisense DNA oligomer treatment of HCT116 cells caused a dose-dependent inhibition of cell growth, there was a subsequent rise in target mRNA product. Because the mechanism of growth inhibition could not involve an inhibition of TGF-alpha expression, it was concluded that Lipofectin probably exerts a nonspecific, detergent-like effect upon the cell membrane, producing an enhancement of TGF-alpha processing and release.

Base Sequence↗
PubMed2001

Expression and localization of LEF-11 in Autographa californica nucleopolyhedrovirus-infected Sf9 cells.

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-11 gene was found previously to be necessary to support optimal levels of transient expression from an AcMNPV late promoter. The lef-11 gene is unusual in that it overlaps both upstream (orf38) and downstream (pp31) genes. In this study, the expression and cellular localization of LEF-11 were examined. The lef-11 transcripts were detected from 4 to 36 h post-infection (p.i.). The 1.5 kb lef-11 mRNA initiates 196 nt upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. This relatively long 5' upstream region encodes a potential small upstream open reading frame (ORF) of 58 amino acids that overlaps the lef-11 ORF. The 3' end of the lef-11 mRNA was mapped as co-terminal with mRNAs from the downstream pp31 gene. Using affinity purified anti-LEF-11 antibodies, levels of LEF-11 expression were found to be maximal between approximately 8 and 24 h p.i., although LEF-11 could be detected as late as 72 h p.i. Using immunofluorescence microscopy, it was determined that LEF-11 localized to dense regions of infected cell nuclei, consistent with its role as a possible late transcription factor.

Amino Acid Sequence↗